We are investigating how heparan sulfate influences FGFR2b signaling in specific progenitor cell types in the epithelium. The exquisite control of growth factor function by HS is dictated by the tremendous structural heterogeneity of its sulfated modifications. It is not known how specific HS structures control growth factor-dependent progenitor expansion during organogenesis. We isolated KIT+ progenitors from fetal salivary glands and profiled HS biosynthetic enzyme expression. Enzymes generating 3-O-sulfated-HS (3-O-HS) are enriched in the KIT+ cells, and FGF10/FGFR2b signaling directly regulates their expression. We used bioengineered 3-O-HS to investigate HS function. 3-O-HS binds FGFR2b and stabilizes FGF10/FGFR2b complexes in a receptor- and growth factor-specific manner. Rapid autocrine feedback increases 3-O-HS, KIT and progenitor expansion. However, the kidney HS used in our previous studies was generated from an undefined GAG chain from bovine kidney that had very low levels of endogenous 3-O-sulfation. We are studying more-defined 3-O-HS derived from cell-lines that do not express Hs3st3s. Secondly, we are testing chemo-enzymatically synthesized HS of defined oligosaccharide size with defined 3-O-HS-sulfation patterns. In both cases, the Hs3st1- and- Hs3st3-treated HS increased endbud morphogenesis of primary fetal salivary epithelium. In addition they increased the expression of genes associated with progenitor cells (Kit), proliferation (Ccnd1), HS synthesis (Hs3st1, Hs3st3a1, Hs3st3b1, Hs3st6, Hs6st1), and Fgfr2b-signaling (Etv5). Defining the minimum saccharide sequences of HS that determine the selectivity and specificity of their function will facilitate the synthesis of small HS mimetics to specifically increase progenitor expansion in vitro. The aim of this project was to identify the source and identity of signals that initiate parasympathetic ganglia (PSG) formation and subsequent organ innervation. Parasympathetic ganglia form near their target organ and innervate the tissue in parallel with development. We hypothesized that a secreted signal from the duct epithelium was critical for neuronal association and subsequent innervation at early stages of SMG development. Microarray analysis of primary duct and endbud epithelia identified the Wnt pathway as the most enriched gene set in the duct. Four Wnts: Wnt4, Wnt5b, Wnt7b, and Wnt10a were more highly expressed in the primary duct and in K5+ progenitors compared to K5- epithelial cells. Using PSG culture, we discovered that Wnts improve PSG neuronal survival, proliferation and ganglion formation. We also showed that excess FGF signaling antagonized Wnt expression in the duct to reduce ganglion formation. To confirm this in vivo, we devised a strategy to reduce expression of multiple Wnts simultaneously by increasing FGF signaling in the epithelium. We did this by genetic deletion of Spry1 and Spry2 (Spry1/2DKO), which are the endogenous inhibitors of FGF signaling in the SMG epithelium. The Spry1/2DKO SMGs had a reduction in the expression of Wnts and as expected, ganglion formation was affected and the PSG was absent, although dispersed neuronal cells were detected in the condensed mesenchyme. Consequently, SMG epithelial development was severely disrupted and K5+ cells were reduced. We treated Spry1/2DKO embryos with a Wnt activator in utero in an attempt to rescue PSG formation, innervation and K5+ progenitors. A single dose of the Wnt activator alone did not improve the phenotype. However, removing a copy of Fgf10 to further decrease FGF signaling (Spry1/2DKO;Fgf10+/-) in combination with the Wnt activator in utero rescued both duct and PSG formation, innervation and K5+ progenitors. In summary, we identified a positive feedback loop where K5+ progenitors produce Wnt signals to promote ganglion formation, leading to gland innervation and K5+ progenitor maintenance. Importantly, this work also highlights the critical need to restrict FGF signaling in the duct to allow Wnt-induced PSG formation and maintenance of K5+ progenitor cells necessary for organogenesis.